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Hybrid Three-Dimensional Lung Tissue Cultures to Improve Ex Vivo Models of Chronic Obstructive Pulmonary Disease

ABSTRACT: Rationale: Continued reliance on preclinical animal models and traditional human cell culture experiments conducted on supraphysiologically stiff surfaces in two dimensions often limits the translation of cellular and molecular mechanistic findings into effective therapies for patients suffering from chronic obstructive pulmonary disease (COPD). COPD ranks as the fourth highest cause of mortality in the United States, and thus it is imperative to improve ex vivo models of this devastating disease. One emerging novel model of study involves using precision-cut lung slices as three-dimensional lung tissue culture (3D-LTC) platforms. Although this model retains the complex pulmonary architecture required for more precise ex vivo modeling, it is limited by short-term viability (?5–7 d). Objectives: We hypothesized that this decrease in viability is related to slices losing the extracellular matrix adhesion foundation critical for normal cellular functioning and that encapsulation of slices in custom hydrogel microenvironments would improve maintenance of a stable phenotype for longer-term investigational studies. Methods: The 3D-LTC slices tested here were created according to standard protocols from healthy mouse lungs and from the lungs of a patient with COPD. We synthesized poly(ethylene glycol)–based polymers to create custom hydrogels for encapsulation of 3D-LTCs with mechanical properties that match each type of lung tissue. The Young’s modulus (E) or stiffness of hydrogels was measured with a parallel-plate rheometer. Lung slices were analyzed for viability with a colorimetric assay that measures cellular metabolism (WST-1; Millipore Sigma) and by staining for cell membrane integrity (Invitrogen LIVE/DEAD viability kit; Thermo Fisher Scientific). All fluorescently labeled samples were imaged by spinning disk confocal microscopy, and cell viability at all time points was analyzed with ImageJ software. Results: The average percent viability of cells within hybrid 3D-LTCs was consistently higher than control 3D-LTCs on Day 1 (35% increase; P?

SUBMITTER: Bailey K

PROVIDER: S-EPMC6322000 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Project description:Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na(+) and K(+) currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for retinal replacement therapies.

Project description:BackgroundDespite their heterogeneity, the current standard preoperative radiotherapy regimen for localized high-grade soft tissue sarcoma (STS) follows a one fits all approach for all STS subtypes. Sarcoma patient-derived three-dimensional cell culture models represent an innovative tool to overcome challenges in clinical research enabling reproducible subtype-specific research on STS. In this pilot study, we present our methodology and preliminary results using STS patient-derived 3D cell cultures that were exposed to different doses of photon and proton radiation. Our aim was: (i) to establish a reproducible method for irradiation of STS patient-derived 3D cell cultures and (ii) to explore the differences in tumor cell viability of two different STS subtypes exposed to increasing doses of photon and proton radiation at different time points.MethodsTwo patient-derived cell cultures of untreated localized high-grade STS (an undifferentiated pleomorphic sarcoma (UPS) and a pleomorphic liposarcoma (PLS)) were exposed to a single fraction of photon or proton irradiation using doses of 0 Gy (sham irradiation), 2 Gy, 4 Gy, 8 Gy and 16 Gy. Cell viability was measured and compared to sham irradiation at two different time points (four and eight days after irradiation).ResultsThe proportion of viable tumor cells four days after photon irradiation for UPS vs. PLS were significantly different with 85% vs. 65% (4 Gy), 80% vs. 50% (8 Gy) and 70% vs. 35% (16 Gy). Proton irradiation led to similar diverging viability curves between UPS vs. PLS four days after irradiation with 90% vs. 75% (4 Gy), 85% vs. 45% (8 Gy) and 80% vs. 35% (16 Gy). Photon and proton radiation displayed only minor differences in cell-killing properties within each cell culture (UPS and PLS). The cell-killing effect of radiation sustained at eight days after irradiation in both cell cultures.ConclusionsPronounced differences in radiosensitivity are evident among UPS and PLS 3D patient-derived sarcoma cell cultures which may reflect the clinical heterogeneity. Photon and proton radiation showed similar dose-dependent cell-killing effectiveness in both 3D cell cultures. Patient-derived 3D STS cell cultures may represent a valuable tool to enable translational studies towards individualized subtype-specific radiotherapy in patients with STS.

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Hybrid Three-Dimensional Lung Tissue Cultures to Improve Ex Vivo Models of Chronic Obstructive Pulmonary Disease

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